Early development of the lung is orchestrated by a small group of critical transcription factors including Gata6, Foxa1/2, and Nkx2.1. Genetic deletion of any of these factors significantly impairs lung endoderm differentiation and in some cases postnatal repair and regeneration. The transcriptional regulatory regions of most lung endoderm restricted genes contain functionally important Gata6, Foxa1/2, and Nkx2.1 DNA binding sites. Gata and Foxa1/2 have been shown to act as "pioneer" transcription factors by binding to regions of compacted chromatin and opening and priming the chromatin landscape to allow subsequent DNA binding by additional factors, resulting in the promotion of cell lineage specific gene expression. Using conditional genetic deletion in lung endoderm, we have demonstrated that Gata6 plays a central role in regulation of lung endoderm gene expression, proliferation, and branching morphogenesis. Loss of Foxa1/2 and Nkx2.1 also dramatically inhibits endoderm differentiation and branching morphogenesis. Moreover, we have demonstrated that Gata6 cooperatively regulates late lung epithelial differentiation and maturation with Nkx2.1. Gata6, Foxa1/2, and Nkx2.1 therefore form a core set of factors that we refer to as a lung restricted transcription regulatory module (TRM). However, how Gata6 cooperates with Foxa1/2 and Nkx2.1 to regulate lung endoderm specific gene expression is poorly understood, particularly in the regulation of early lung endoderm progenitor specification and differentiation. Recent unpublished data from our lab suggests that Gata6 controls lung epithelial differentiation and proliferation, in part, though regulation of the microRNA cluster miR-302/367. miR-302/367 is expressed in early embryonic development in multiple stem/progenitor cell populations and gain of function studies in vivo shows that it regulates proliferation and differentiation of early lung endoderm progenitors. Our unifying hypothesis is that Gata6 acts as an integrator of early lung endoderm progenitor development through interactions with Foxa1/2 and Nkx2.1. These interactions, in turn, regulate critical down-stream targets in lung endoderm including the microRNA cluster miR-302/367. To test this hypothesis, we propose to 1) characterize the regulation of the miR-302/367 cluster by Gata6, Foxa1/2, and Nkx2.1, 2) determine the role of miR-302/367 in lung endoderm progenitor development, and 3) determine how Gata6, Foxa1/2, and Nkx2.1 integrate regulation of early lung development and the lung endoderm transriptome. PUBLIC HEALTH RELEVANCE: A better understanding of the molecular mechanisms that regulate early lung development is directly relevant to both congenital lung disease as well as potential lung regenerative therapies. A core set of transcriptional regulators consisting of Gata6, Foxa1/2, and Nkx2.1 regulate a majority of lung specific genes and are important for adult lung homeostasis and injury repair. Thus, the studies described in our proposal will greatly enhance our knowledge of the pathways that regulate early lung progenitor development as well as airway regeneration in the adult.